Combination of acid protease enzymes and acidic buffers and uses thereof

ABSTRACT

Novel compositions comprising one or more of an acid protease and an acidic buffer, the acidic buffer comprising an acid and a pharmaceutically or cosmetically 5 acceptable carrier, vehicle or excipient, useful for treating or preventing abnormal biological conditions, diseases or disorders, and/or for improving the texture or appearance of the skin, and/or for enhancing epidermal exfoliation and/or for enhancing epidermal cell renewal and to methods for the use of the compositions. The acid protease comprises one or more proteolytic enzymes that exhibit proteolytic activity at pH values below that of the surface of the skin, i.e., approximately pH 5.5. The acidic buffer comprises inorganic and/or organic acids or mixtures thereof with a pharmaceutically or cosmetically acceptable carrier, vehicle or excipient. The buffer is capable of reducing the pH of the surface of the skin to less than pH 5.5 and is susceptible to neutralization by normal epidermal processes.

This is a divisional of application Ser. No. 08/664,056, filed Jun. 13,1996, now U.S. Pat. No. 5,976,556.

TABLE OF CONTENTS

1.0 FIELD OF THE INVENTION

2.0 BACKGROUND OF THE INVENTION

3.0 SUMMARY OF THE INVENTION

3.1 DEFINITIONS

4.0 BRIEF DESCRIPTION OF THE DRAWINGS

5.0 DETAILED DESCRIPTION OF THE INVENTION

5.1 COMPOSITIONS

5.2 METHODS OF USE OF THE COMPOSITIONS OF THE PRESENT INVENTION

6.0 EXAMPLES

6.1 ENHANCEMENT OF SKIN EXFOLIATION

6.2 COMPOSITIONS FOR USE IN THE METHODS OF THE PRESENT INVENTION

6.3 STABILIZATION COMPOSITION

6.4 PROTEOLYTIC ACTIVITY AT VARYING pH LEVELS

1. FIELD OF THE INVENTION

This invention relates to novel compositions comprising one or more ofan acid protease enzyme and an acidic buffer, the acidic buffercomprising an acid and a pharmaceutically or cosmetically acceptablecarrier, useful for treating or preventing abnormal skin conditions,diseases or disorders, and/or for improving the texture or appearance ofthe skin, and/or for enhancing epidermal exfoliation, and/or forenhancing epidermal cell renewal and to methods for the use of thecompositions.

2. BACKGROUND OF THE INVENTION

It is well founded that exfoliation of epidermal layers of human skininduces an increased rate of epidermal cell renewal (E. Phillips, 1995,U.S. Pat. No. 5,431,913; W. P. Smith, 1994, Cosmetics and Toiletries109:41-8). The human epidermis consists of multiple layers of stratifiedsquamous epithelial cells in a constant state of renewal. New cells areformed first in the basal layer, which is the most internal membrane ofthe epidermis. These cells are displaced by the production of yet newercells and subsequently are transported to the external layer of theepidermis, the stratum corneum, where they usually are shed (exfoliated)every two to three weeks. The general health and appearance of humanskin depends greatly upon the rate of this process.

Certain situations or conditions, such as aging or exposure to theenvironment, can disturb this normal process and can lead to a generallyreduced rate of cell renewal as well as an increased degree ofintercorneocyte cohesion (VanScott and Yu, 1984, J. Am. Acad. Dermatol.11:867-79). Because the time required for a cellular layer to migratefrom the basal layer to the stratum corneum increases with the subject'sage, the rate of epidermal cell renewal decreases. It has been reportedthat in a typical twenty year old person, the cells in the outer layersof the epidermis turn over on the average, every two weeks, while cellturnover intervals of more mature skin can be as much as twice as long.(E. Phillips, 1995, U.S. Pat. No. 5,431,913). The decrease in cellrenewal rate due to aging can be exacerbated by environmental conditionssuch as exposure to solar radiation and other climatic conditions (K. E.Burke, 1990, Postgraduate Medicine 88(1):207-27). A decrease in the cellrenewal rate increases the time cells in the outer layers of skin areexposed to environmental conditions and may lead to further and/orcumulative damage. Certain studies suggest that a decrease in epidermalcell turnover rates is associated with an increase in intercorneocytecohesion (VanScott and Yu, 1989, Cutis 43:222-28).

The cells of the epidermal layer are held together by proteinaceouscomponents such as hemidesmosomes, desmosomes, gap junctions,glycosaminoglycans, proteoglycans, and other components present in theskin all of which have the ability to bind to each other and to cellularcomponents. This binding together of the epidermal cells is described as“intercorneocyte cohesion”, and the degree of the cohesive strengthinvolved is affected by such factors as hydration and pH. Increasedintercorneocyte cohesion results in hyperkeratinization and ischaracterized by thick and often dry or scaly skin caused by theretention of epidermal cells. The balance that exists between cohesionand cell turnover rates is responsible for normal rejuvenation of youngskin and imbalances of these factors can result in the aged, rough, andunattractive look of mature epidermal surfaces. The search for topicallyactive components that balance cell renewal rates and intercorneocytecohesion is prominent in dermatological research efforts today (VanScottand Yu, 1984, J. Am. Acad. Dermatol. 11:867-79).

Recent advances in this research area have provided a number oftopically active acidic compounds which show promise in this regard (Yuet al., 1978, U.S. Pat. No. 4,105,783; Yu et al., 1982, U.S. Pat. No.4,363,815). Long term treatments with these acidic compounds result inan increased rate of epidermal cell renewal. Those acidic componentsknown to be epidermally active, such as low molecular weight hydroxy orketo acids and esters thereof, are well documented with respect to theireffectiveness as keratolytic/desquamation agents. High concentrations ofthese acids (e.g., salicylic and glycolic acids), as well as other acidssuch as trichloroacetic acid, are known for their ability to causedestruction of tissue at the site of application (W. L. Epstein, 1990,in Irritant Contact Dermatitis, Jackson and Glodner, eds., MarcelDecker, Inc., New York and Basel, pp. 127-165; Remington'sPharmaceutical Sciences, A. Osol, ed., Sixteenth Ed., Mack Publishing,Inc., Easton, Pa., 1980). At lower concentrations, these acids have beenshown to have the ability to loosen the dead cells in the keratin-richstratum corneum and interrupt intercorneocyte cohesion therebyfacilitating desquamation; this activity is called “keratolyticactivity” (Remington's Pharmaceutical Sciences, A. Osol, ed., SixteenthEd., Mack Publishing, Inc., Easton, Pa., 1980; W. P. Smith, 1994,Cosmetics and Toiletries 109:41-8).

Most keratolytics are skin irritants, including those listed above.Although some keratolytics are promoted as being more efficient thanothers, a comparison of the therapeutic index of each of these acidsshows little advantage of one over another. The “therapeutic index” isan accurate method of rating the efficiency of these components whichtakes into account the component's keratolytic efficiency as well as thelevels of irritation at a given concentration. It has been shown thateven low levels of these components (greater than 4% acid concentration)cause significant skin irritation. The use of even lower levels of thesecomponents reduces irritation but generally reduces keratolyticeffectiveness. Furthermore, prolonged use of the acids reduces theefficiency of cell renewal induction (W. P. Smith, 1994, Cosmetics andToiletries 109:41-8). These shortcomings point out the need for amethodology to enhance the keratolytic effectiveness of these acids atnon-irritating concentrations.

The application of proteolytic enzymes in topical therapy has been usedfor some time for such things as scar removal from burn wounds and as anadjunct to anti-microbial therapy (G. Rodeheaver, 1975, Am. J. Surg.129(5):537-544). These enzymes include those generally restricted toplant sources, such as papaya (papain), fig (ficin), and pineapple(bromelain). Cosmetic formulations containing extracts from these plantsand promoted as enhancing the removal of outer epidermal layers byproteolytic action have been marketed commercially. Although thesubstrate specificity and spectrum of activity in relation to pH suggestthat the proteolytic action of these formulations should result inkeratolytic activity at the outer epidermal layers, applications ofproteolytic enzymes previously used in topical therapy have not beenwithout drawbacks.

Those enzymes (e.g., papain, bromelain, and ficin) currently used incosmetic applications generally exhibit proteolytic activity over abroad pH range of pH 3-pH 9 (Glazer and Smith, 1971, in The Enzymes,Vol. 3, P. Boyer, ed., Academic Press, New York, pp. 501-546). It isprobably the broad pH range of these plant-derived enzymes that isresponsible for at least some of the problems associated with prolongedskin exposure. Human epidermal systems maintain a pH gradient within thestratified layers of the skin; the outer layers have been reported toexhibit an average pH of 5.5 (W. P. Smith, 1994, Cosmetics andToiletries 109:41-48). The pH of successive layers of the epidermisincreases with depth, reaching a final pH closer to the physiologicalrange (about pH 7.4) at the dermal layer. Because the plant-derivedenzymes mentioned above remain active across this pH gradient, there isno pH control over these plant enzyme activities when they are appliedto the skin.

The degree of therapeutic activity received by topical application ofproteolytic enzymes has been governed by the intrinsic catalyticcharacteristics of those enzymes. The wide range of proteolyticactivity, in relation to pH, exhibited by the proteases discussed above(pH 3-9) allows for little or no control over proteolytic activity byproduct. formulation (Glazer and Smith, 1971, in The Enzymes, Vol. 3, P.Boyer, ed., Academic Press, New York, pp. 501-546). The plants fromwhich these enzymes have been obtained are known to cause irritantcontact dermatitis with symptoms including itching, edema, andblistering. It has been postulated that these enzymes are the primarycause of those symptoms possibly due to excessive decreases inintercorneocyte cohesion (W. L. Epstein, 1990, in Irritant ContactDermatitis, Jackson and Glodner, eds., Marcel Decker, Inc., New York andBasel, pp. 127-165). The enzymes currently used in cosmetic applicationscan exhibit substrate-dependent proteolytic activity throughout alllayers of the epidermis. Uncontrolled proteolysis of epidermal proteinsthat serve to stabilize intercorneocyte cohesion could decrease thisintercorneocyte cohesion excessively and cause subsequent irritation.Proteolytic attack at the basal layer of the epidermis, where cellrenewal originates, could cause imbalances in the rate of epidermal cellrenewal. Blistering and edema could be the result of uncontrolledproteolytic attack by this class of enzymes at the basal membrane. Thispossibility is supported by the fact that injection of these plantenzymes into the skin of mammals is known to cause edema in the skin(Morimoto et al., 1987, Ensho 7(6):563-567). This finding has been usedto produce experimental edema in dermatological research. Furthermore,the symptoms of irritant contact dermatitis listed above are similar tothose induced by the most effective concentrations of epidermally activeacidic components currently used in topical applications. Therefore, amore controlled approach to proteolytic activity with respect to topicalapplications should produce a desirable result without these sideeffects.

Compositions containing a proteolytic enzyme, pepsin, and several acidshave been reported for the dissolution of dense necrotic formationsseen, for example, with third degree burns (Soviet Union Patent No.439288, 1974). There is no indication that these compositions are usefulin enhancing epidermal exfoliation or that these compositions are activeonly for limited time periods.

Citation or identification of any reference in the background of thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention.

3. SUMMARY OF THE INVENTION

The present invention provides novel pharmaceutical and/or cosmeticcompositions for treating or preventing abnormal skin conditions,diseases or disorders, and/or for improving the texture or appearance ofthe skin, and/or for enhancing epidermal exfoliation, and/or forenhancing epidermal cell renewal. The compositions comprise one or moreof an acid protease and an acidic buffer. For purposes of thisinvention, the acid protease is an enzyme which exhibits peptidylhydrolase (proteolytic) activity below the average pH of the surface ofthe skin and is significantly inactive at a pH greater than the averagepH of the surface of the skin, which is about pH 5.5 for humans (W. P.Smith, 1994, Cosmetics and Toiletries 109:41-8). For men the average pHof the surface of the skin is about pH 5.3 and for women it is about pH6.0 (Ohman and Vahlquist, 1994, Acta Dermato-Venereol. 74(5):375-379).For purposes of this invention, the average pH of the surface of theskin which is about pH 5.5 includes, in addition to others,gender-specific variations, such that a pH of about 5.5 includes a rangeof about pH 5.3, the average pH of the surface of a man's skin, up toabout pH 6.0, the average pH of the surface of a woman's skin.Preferably, at a pH greater than or equal to the average pH of thesurface of the skin (approximately pH 5.3-6.0), the acid proteasesuseful in this invention exhibit less than about 10% of the enzymaticactivity they exhibit at their respective optimal pHs below the averagepH of the surface of the skin.

The acid protease can be in the apoenzyme, holoenzyme, isoenzyme, orzymogen form. The acid protease component of the composition can bepresent in an amount of about 0.001% to about 75.0% by weight of thefinal composition, preferably about 0.1% to about 50%, more preferablyabout 1% to about 5%. The protease(s) has a specific activity of about1.0 to about 5,000 HUT units/mg as determined by the method, asmodified, described in Food Chemicals CODEX, 3rd ed., (1981), pp.496-497, National Academy Press, Washington, D.C., see infra, Section5.1. Preferably the protease(s) has a specific activity of about 50 toabout 3000 HUT units/mg, more preferably about 500 to about 1500 HUTunits/mg.

The acidic buffer is a composition which when topically applied to theskin, temporarily lowers the pH of the surface of the skin to less thanabout pH 5.5 but not lower than about pH 1.0, preferably to betweenabout pH 2.5 and about pH 4.5. The acidic buffer composition comprisesat least one acid and a pharmaceutically or cosmetically acceptablecarrier, vehicle or excipient. The acid component of the buffer can bean inorganic or an organic acid or mixtures thereof. The acidic bufferis susceptible to neutralization to the average pH of the surface of theskin over time by natural epidermal processes, such as perspiration. Thetime period required for neutralization, and subsequent inactivation ofthe protease, will depend on the formulation of the acidic buffer. Forexample, shorter time periods result if the acidic buffer contains aweak acid or a weak buffering agent to counteract the relativealkalinity of the epidermis; longer time periods result if a strongeracid is utilized or a stronger buffering agent is employed in the acidicbuffer. The acid component of the acidic buffer of the composition canbe an organic acid or an inorganic acid or mixtures thereof and can bepresent in an amount of about 0.001% to about 95.0% by weight of thefinal composition, preferably about 0.01% to about 25%, more preferablyabout 1.0% to about 5%.

Control of the time period required for the pH of the surface of theskin to return to a pH of about 5.5 after topical application of acomposition of the present invention allows for control of the activityof the protease enzyme. It is through this control of proteolyticactivity that the present invention overcomes the drawbacks andcomplications found in the prior art, such as itching, burning,blistering, etc., caused by broad pH spectrum proteolytic enzymes. Theperiod of time it takes for the surface pH of the skin to return toabout pH 5.5 is determined by a number of factors, including the type ofskin condition, disease or disorder that is being treated and thesensitivity of the skin of the particular subject being treated. Toavoid the drawbacks and complications found in the prior art, ideally,the period of time should not exceed about 4 hours for any individualapplication of a composition of the present invention, preferably theperiod of time is between about minutes to about 4 hours, morepreferably between about minutes to about 2 hours, most preferablybetween about minutes to about one hour.

It is to be pointed out that the pharmaceutical compositions of thepresent invention are those which, when administered to the skin, rendera benefit or an effect of treating or preventing an abnormal biologicalcondition, disease, or disorder. Benefits or effects of treating orpreventing such abnormal condition, disease, or disorder are thereduction in severity or disappearance of the symptoms or cause of theabnormal condition, disease, or disorder. The reduction in severity ordisappearance of the abnormal condition, disease, or disorder may beeither in the short-or long-term. Such abnormal biological conditions,diseases, or disorders to be treated by administering a composition ofthe present invention include, but are not limited to, dry skin, severedry skin, dandruff, acne, keratoses, eczema, skin flakiness, pruritus,age spots, lentigines, melasmas, wrinkles (both coarse and fine, causedby intrinsic as well as extrinsic damage), warts, blemished skin,hyperpigmented skin, hyperkeratotic skin, inflammatory dermatoses,age-related skin changes and skin in need of cleansers, as well as theeffects of skin atrophy and psoriasis.

It is to be further pointed out that the cosmetic compositions of thepresent invention are those which, when administered to the skin,improve the texture or appearance thereof or enhance epidermalexfoliation and/or epidermal cell renewal, without necessarily renderinga benefit or an effect of treating or preventing an abnormal biologicalcondition, disease, or disorder. In this context, improving the textureor appearance of the skin or enhancing epidermal exfoliation and/orepidermal cell renewal is meant to encompass providing a natural-lookingand/or natural-feeling coating over the skin so as to enhance the beautyand/or smoothness of the skin from its pre-treated state, or to maskunwanted symptoms of an abnormal biological condition, disease, ordisorder. This can include providing a temporary moisturizing effect tothe epidermis. Such abnormal biological conditions, or diseases include,but are not limited to dry skin, severe dry skin, dandruff, acne,keratoses, psoriasis, eczema, skin flakiness, pruritus, age spots,lentigines, melasmas, wrinkles (both coarse and fine, caused byintrinsic as well as extrinsic damage), warts, blemished skin,hyperpigmented skin, hyperkeratotic skin, inflammatory dermatoses,age-related skin changes and skin in need of cleansers, as well as theeffects of skin atrophy, and psoriasis.

In yet another embodiment, the invention encompasses methods fortreating abnormal skin conditions, diseases, or disorders including butnot limited to dry skin, severe dry skin, dandruff, acne, keratoses,psoriasis, eczema, skin flakiness, pruritus, age spots, lentigines,melasmas, wrinkles (both coarse and fine, caused by intrinsic as well asextrinsic damage), warts, blemished skin, hyperpigmented skin,hyperkeratotic skin, inflammatory dermatoses, age-related skin changesand skin in need of cleansers. The method comprises topicallyadministering to an area of a subject's skin having the condition,disease, or disorder an effective a composition comprising an acidprotease which is enzymatically active below about pH 5.5 and an acidicbuffer which lowers the surface pH of the skin to below about pH 5.5 fora period of time effective for the treatment of the condition, disease,or disorder, the acidic buffer being subject to neutralization by normalepidermal processes.

In yet another embodiment, the present invention provides methods forthe enhancement of epidermal exfoliation and/or epidermal cell renewalcomprising topically administering to an area of a subject's skin aneffective amount of a composition comprising an acid protease which isenzymatically active below about pH 5.5 and an acidic buffer whichlowers the surface pH of the skin to below about pH 5.5 for a period oftime effective for enhancing epidermal exfoliation and/or epidermal cellrenewal, the acidic buffer being subject to neutralization by normalepidermal processes.

In another embodiment, the invention provides methods for improving thetexture or appearance of the skin comprising topically administering toan area of a subject's skin an effective amount of a compositioncomprising an acid protease which is enzymatically active below about pH5.5 and an acidic buffer which lowers the surface pH of the skin tobelow about pH 5.5 for a period of time effective to improve the textureor appearance of the skin, the acidic buffer being subject toneutralization by normal epidermal processes.

In a still further embodiment, the invention provides methods forregulating the effects of skin atrophy comprising administering to anarea of a subject's skin an effective amount of a composition comprisingan acid protease which is enzymatically active below about pH 5.5 and anacidic buffer which lowers the surface pH of the skin to below about pH5.5 for a period of-time effective for regulating the effects of skinatrophy, the acidic buffer being subject to neutralization by normalepidermal processes.

In a preferred embodiment for enhancing epidermal exfoliation and/orepidermal cell renewal, the composition comprises pepsin and lacticacid. In this preferred embodiment for enhancing epidermal exfoliationand/or cell renewal, the composition comprises about 1.0% by weight ofpepsin having a specific activity of about 1000 HUT units/mg and about3.0% by weight of lactic acid. In a another preferred embodiment forenhancing epidermal exfoliation and/or cell renewal, the compositioncomprises about 1.0% by weight of pepsin having a specific activity ofabout 1000 HUT units/mg and about 1.5% by weight of lactic acid. In yetanother preferred embodiment the composition comprises 1.0% by weight ofpepsin and 1.0% by weight phosphoric acid. In another preferredembodiment the composition comprises 1.0% by weight of pepsin and 3.0%by weight phosphoric acid.

The compositions and methods of the present invention surprisinglydemonstrate pharmaceutical activity or cosmetic effects against skindisorders heretofore not achieved by acids, α-hydroxycarboxylic acids,salicylic acids or broad pH spectrum proteases by themselves.

3.1 DEFINITIONS

As used in the present invention the following terms are intended toencompass the following:

ACIDIC BUFFER: A composition comprising an acid and a pharmaceuticallyor cosmetically acceptable carrier, vehicle or excipient which whentopically applied to the skin lowers the surface pH of the skin to belowabout pH 5.5 and is subject to neutralization by natural skin processessuch that the natural skin processes, over time, return the pH of theskin's surface to normal, which is about pH5.5. The acid component ofthe buffer can be either an inorganic acid or an organic acid ormixtures thereof.

ACID PROTEASE: An enzyme which exhibits peptidyl hydrolase (proteolytic)activity at a pH below the average normal pH of the skin's surface,which is about pH 5.5, and which is significantly inactive at a pHgreater than or equal to such average normal skin surface pH, i.e., lessthan about 10% activity at about pH 5.5 or greater as compared to peakactivity at pH less than about pH 5.5. The acid protease can be in theapoenzyme, holoenzyme, isoenzyme or zymogen form.

COSMETIC: A formulation to be administered to the skin which improvesthe texture or appearance thereof, without necessarily rendering abenefit or an effect of treating or preventing an abnormal biologicalcondition or a disease. Such improvement includes providing a temporarymoisturizing effect to the mammalian epidermis.

EFFECTIVE AMOUNT:

An amount of composition sufficient to significantly induce a positivemodification in the condition to be treated, but low enough to avoidserious side effects. The effective amount of the composition will varywith the particular condition being treated, the age and physicalcondition of the subject being treated, the severity of the condition,the duration of the treatment, the nature of concurrent therapy, thespecific composition employed, the particularpharmaceutically-acceptable carrier or cosmetically-acceptable carrierutilized, and similar factors within the knowledge and expertise ofthose skilled in the art.

EPIDERMAL CELL RENEWAL:

The process by which new skin cells are formed in the basal layer, aretransported to the external layer, the stratum corneum, and subsequentlyare exfoliated and replaced by yet newer skin cells.

EXFOLIATION:

The detachment and shedding of superficial cells of an epithelium orfrom any tissue surface.

PHARMACEUTICAL:

A formulation to be administered to the skin which renders a benefit oran effect of treating or preventing an abnormal biological condition ora disease.

REGULATING SKIN ATROPHY:

The preventing, retarding, arresting, treating, or reversing the processof atrophy in mammalian skin.

SKIN ATROPHY: The thinning and/or general degradation of the dermislayer of mammalian skin often characterized by a decrease in collagenand/or elastin as well as decreased number, size and doubling potentialof fibroblast cells. Skin atrophy is a natural result of aging, but maybe caused by either intrinsic or extrinsic factors such as naturalchronoaging, photodamage, burns or chemical damage, or by exposure topollutants or allergens, e.g., cigarette smoke. Skin atrophy is often anundesirable side effect resulting from treatment withα-hydroxycarboxylic acids or salicylic acids.

The present invention may be understood more fully by reference to thedetailed description and illustrative examples which are intended toexemplify non-limiting embodiment e invention.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph demonstrating the effect on the rate of cellrenewal with different concentrations of an acid protease (pepsin) withtwo different amounts of an acidic buffer (lactic acid). ▪ 1.5% lacticacid, □ 3.0% lactic acid.

FIG. 2 is a bar graph demonstrating the percent increases inexfoliation/cell renewal rates given by various concentrations of anacid protease (pepsin) over that of an acidic buffer alone (lactic acid)at concentrations of 1.5% and 3.0%. ▪ 1.5% lactic acid, □ 3.0% lacticacid.

FIG. 3 is a bar graph demonstrating the difference in activity of pepsinand AFP 2000, Solvay, Inc., Elkhart, Ind., a fungally-derived acidprotease, at varying pH levels in the HUT assay. ▪ AFP 2000, pepsin.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1 COMPOSITIONS

The present invention provides novel compositions for treating orpreventing abnormal skin conditions, diseases or disorders, and/or forimproving the texture or appearance of the skin, and/or for enhancingepidermal exfoliation, and/or for enhancing epidermal cell renewal. Thecompositions comprise one or more of an acid protease and an acidicbuffer. Such compositions are preferably to be administered topically,so as to minimize systemic effects or undesirable side effects. Theinventor does not wish to be limited by a specific mode of action;however, the inventor believes that the compositions of the presentinvention accomplish the methods of the present invention by firstlowering the pH of the surface of the skin to a value lower than theaverage pH of the skin, which is about 5.5 for humans. The reduction inpH allows for the activation of the acid protease component of thecomposition which exhibits proteolytic/keratolytic activity during thetime that the skin's surface pH is below about 5.5. This time perioddepends upon the skin pH conditions of the individual and can beregulated by the formulation of the acidic buffer component. It iswithin the skill of the art to adjust the formulation so as to vary thetime period and achieve the desired result.

During this time period, the acidic buffer is neutralized by naturalepidermal processes, such as perspiration, such that the pH of theskin's surface returns to normal, which averages about pH 5.5. Theincrease in pH thus inactivates the acid protease andproteolytic/keratolytic activity ceases. The period of time in which thepH of the skin's surface is below about pH 5.5 and in which the proteasecomponent is active, is between about 1 minute to about 4 hours,preferably between about minutes to about 2 hours, more preferablybetween about 30 minutes to about 1 hour.

For purposes of this invention, the acid protease is an enzyme whichexhibits significant peptidyl hydrolase (proteolytic) activity belowabout pH 5.5, which is the average normal pH of the surface of the skin,and which is significantly inactive at a pH of about 5.5 or greater. Formen the average pH of the surface of the skin is about pH 5.3 and forwomen it is about pH 6.0. For purposes of this invention, the average pHof the surface of the skin which is about pH 5.5 includes, in additionto others, gender-specific variations, such that a pH of about 5.5includes a range of about pH 5.3, the average pH of the surface of aman's skin, up to about pH 6.0, the average pH of the surface of awoman's skin. The protease is significantly inactive when its activityis equal to or less than 10% activity as compared, for example, to itsactivity shortly after being applied to the skin as measured by thedansyl chloride test described in Section 6.1. Acid proteases useful inthe present invention are those which, in an in vitro assay at pHs ofabout 5.5 and greater, display less than about 10% of the enzymaticactivity measured in the same in vitro assay at the optimum pH (lessthan about pH 5.5) of the particular protease. See, e.g., Section 6.4,infra.

The acid protease can be in the apoenzyme, holoenzyme, isoenzyme, orzymogen form. Further, the acid proteases can be isolated from anysource known to those of skill in the art, such as, for example,bacteria, fungi, tissue culture cells, and animals. The acid proteasepreparation can be present in an amount of about 0.001% to about 75.0%by weight of the final composition, preferably about 0.1% to about 50%,and most preferably about 1.0% to about 5.0%. The protease(s) will havea specific activity of about 1 to about 5000 HUT units/mg, preferablyabout 50 to about 3000 HUT units/mg, and most preferably about 500 toabout 1500 HUT units/mg as determined by the method described in FoodChemicals CODEX, 3rd ed., (1981), pp. 496-497, National Academy Press,Washington, D.C., as modified described below.

Briefly, one first prepares the following stock solutions: HEMOGLOBINSUBSTRATE is prepared by mixing 2 g bovine hemoglobin into 80 mldistilled water. The solution is then titrated to pH 2 by adding, forexample, phosphoric acid and/or citric acid and additional distilledwater is added for a total volume of 100 ml. This solution is separatedinto four equal portions and each portion is titrated to the desired pHwith 50% sodium hydroxide or 50% hydrochloric acid. These finalsolutions are then heated at 30° C. for 20 minutes and are then filteredthrough glass wool. TCA STOCK is prepared by dissolving trichloroaceticacid in distilled water for a final concentration of 5% TCA.

Next, for each sample for which proteolytic activity is to be measured,prepare tubes labeled “B” and “T”. In each tube place 4 ml of thehemoglobin solution and place at 37° C. such that the sample isprewarmed to 37° C. Into the “T” tube, add 100 μg enzyme solution, swirlgently and incubate at 37° C. for minutes. Next add ml TCA stocksolution to each tube. Add an equal amount of the enzyme as above addedto the “T” tube to the tube marked “B” which is the control forbackground absorbance. Centrifuge each tube and filter each samplethrough a syringe filter and place the filtered sample into a quartzcuvette for reading the absorbance at 280 nm. The actual absorbance isdetermined by subtracting the “T” sample's absorbance from thebackground. A standard curve can be generated by measuring knownquantities of protease.

One HUT unit of proteolytic activity is defined as that amount of enzymethat produces, in one minute under the specified conditions of theassay, a hydrolysate whose absorbance at 280 nm is the same as that of asolution containing 1.10 μg per ml of tyrosine in 0.006 N hydrochloricacid. HUT units per gram are determined by the following formula:

HUT/g=(absorbance at 280 nm×V)/(0.0084×T×W)

where V is the final volume of the test solution, T is the reaction timein minutes, and W is the dry weight of the original enzyme sample usedin the assay in grams. Protein concentration is determined by any methodknown in the art, such as, for example, the Bradford Assay which isdescribed in Current Protocols in Molecular Biology, Ausubel et al.,(Eds.), John Wiley & Sons, Inc., New York, 1994.

Examples of such proteases include, but are not limited to, pepsin,cathepsin, human urinary acid protease, fungal proteases derived fromNeurospora oryzae, Mucor pusillus, Mucor miehei, Rhizopus chinensis, orEndothia parasitica, bacterial proteases rhizopuspepsin,penicillopepsin, and endothiapepsin. Further, the proteases may bederived from processes involving genetic engineering processes andtechniques, whether involving embryonic, mature, or induced cells andproducts including DNA segments, plasmids, vectors, or expressionthereof, or transformed cells, or pure cultures. It should be understoodthat two or more acid proteases can be used in combination such that thecombined amount in weight percent is within the ranges mentioned above.

The acidic buffer is a composition which when topically applied to theskin, temporarily lowers the pH of the surface of the skin to less thanabout pH 5.5, but not lower than about pH 1.0, preferably about pH 2.5to about pH 4.5. The acidic buffer composition comprises as least oneacid and a pharmaceutically or cosmetically acceptable carrier, vehicle,or excipient. The acid component of the buffer is susceptible toneutralization to the average normal pH of the surface of the skin overtime by natural epidermal processes, for example, by perspiration. Thetime period required for neutralization, and subsequent inactivation ofthe protease, will depend on the formulation of the acidic buffer. Forexample shorter time periods result if the acid and/or buffering agentin the acidic buffer are weak relative to the neutralizing capacity ofthe epidermis; longer time periods result if a stronger acid is utilizedor a stronger buffering agent is employed in the acidic buffer. The acidcomponent of the acidic buffer of the composition can be an organic acidor an inorganic acid or mixtures thereof and can be present in an amountof about 0.001% to about 95.0% by weight of the final composition,preferably about 0.01% to about 25%, more preferably about 1.0% to about5.0%. The weight % of the acid component will depend on acid strengthand molarity. Moreover, the amount and strength of the acid component inthe composition should be such that effective levels ofproteolytic/keratolytic activity occurs but significant levels of skinirritation do not. Further, the acid component of the acidic buffer mayor may not comprise an acid which has not been shown to exhibit anykeratolytic activity but which merely provides an acidic environment forthe activation of the protease. Examples of such acids include anymolecule that can be manipulated in any composition to buffer theepidermal pH at pH ranges from about 0.1 to about 7 including, but arenot limited to, lactic acid, sorbic acid, phosphoric acid, citric acid,glycolic acid, malic acid, gluconic acid, pyrophosphoric acid,triphosphoric acid, polyphosphoric acid, sodium bisulfate and potassiumbisulfate. It should be understood that two or more acids can be used incombination such that the combined amount in weight percent is withinthe ranges mentioned above.

The acidic buffer also contains a component which is a pharmaceuticallyor cosmetically acceptable carrier, vehicle, or excipient. Examples ofsuch pharmaceutically acceptable carriers, vehicles, or excipients arewell known to those skilled in the art and can be found, for example, inRemington's Pharmaceutical Sciences, Eighteenth Edition, A. R. Gennaro,Ed., Mack Publishing Co., Easton, Pa., 1990. Examples of suchcosmetically acceptable carriers or excipients are well known to thoseskilled in the art and can be found, for example, in CTFA InternationalCosmetic Ingredient Dictionary, Fourth Edition, J. M. Nikitakis, Ed.,The Cosmetic, Toiletry, and Fragrance Association, Washington, D.C.,1991.

The compositions of the present invention intended for topicalapplication may contain carrier, excipient, or vehicle ingredients suchas, for example, water, acetone, ethanol, ethylene glycol, propyleneglycol, butane-1,3-diol, isopropyl myristate, isopropyl palmitate,mineral oil, and mixtures thereof to form lotions, tinctures, creams,emulsions, gels, or ointments which are non-toxic and pharmaceutically,cosmetically, or dermatologically acceptable. Additionally, moisturizersor humectants can be added to the present compositions if desired.Examples of such additional ingredients useful for pharmaceuticalcompositions can be found in Remington's Pharmaceutical Sciences,Eighteenth Edition, A. R. Gennaro, Ed., Mack Publishing Co., Easton,Pa., 1990. Examples of such additional ingredients useful for cosmeticcompositions can be found in CTFA International Cosmetic IngredientDictionary, Fourth Edition, J. M. Nikitakis, Ed., The Cosmetic,Toiletry, and Fragrance Association, Washington, D.C., 1991.

In addition to these and other vehicles which will be obvious to thoseof ordinary skill in the art, it will be understood that thepharmaceutical and cosmetic compositions of the present invention mayinclude other ingredients such as, for example and not by way oflimitation, those that improve or eradicate age spots, keratoses, andwrinkles; analgesics; anesthetics; antiacne agents; antibacterials;antiyeast agents; antifungal agents; antiviral agents; antidandruffagents; antidermatitis agents; antipruritic agents; antiemetics;antimotion sickness agents; antiinflammatory agents;antihyperkeratolytic agents; antidryskin agents; antiperspirants;antipsoriatic agents; antiseborrheic agents; hair conditioners and hairtreatment agents; antiaging and antiwrinkle agents; antiasthmatic agentsand bronchodilators; sunscreen agents; antihistamine agents; skinlightening agents; depigmenting agents; vitamins; corticosteroids;tanning agents; hormones; retinoids; topical cardiovascular agents;clotrimazole; ketoconazole; miconazole; griseofulvin; hydroxyzine;diphenhydramine; pramoxine; lidocaine; procaine; mepivacaine;monobenzone; erythidocaine; procaine; mepivacaine; monobenzone;erythromycin; tetracycline; clindamycin; meclocyline; hydroquinone;minocycline; naproxen; ibuprofen; theophylline; cromolyn; albuterol;retinoic acid; 13-cis retinoic acid; hydrocortisone; hydrocortisone21-acetate; hydro-cortisone 17-valerate; hydrocortisone 17-butyrate;betamethasone valerate; betamethasone dipropionate; triamcinoloneacetonide; fluocinonide; clobetasol propionate; benzoyl peroxide;crotamiton; propranolol; promethazine; vitamin A palmitate; vitamin Eacetate hydrophilic thickening agents used in pharmaceuticalformulations and mixtures thereof. Concentrations of these ingredientswill vary depending upon intended use, e.g., therapeutic or cosmetic.

Control of the time period required for the pH of the surface of theskin to return to a normal pH of about 5.5 after topical application ofa composition of the present invention allows for control of theactivity of the protease enzyme. It is through this control ofproteolytic activity that the present invention overcomes the drawbacksand complications found in the prior art, such as itching, burning,blistering, and other undesirable side effects caused by broad pHspectrum proteolytic enzymes. The determination of the period of time ittakes for the surface of the skin to return to a normal pH of about pH5.5 is determined by a number of factors, including the type of skincondition, disease, or disorder that is being treated, the type of skinand its location on the body, and the sensitivity of the skin of theparticular subject being treated. To avoid the drawbacks andcomplications found in the prior art the period of time ideally shouldnot exceed about 4 hours for any individual application of a compositionof the present invention. The time limit will depend on a number offactors, such as the purpose of the application, for example, day to daymaintenance, removal of rough dry skin, or therapeutic/pharmaceuticalindications. Other factors include skin type, whether its sensitive,normal, insensitive, damaged or severely damaged and the mode ofapplication, for example, a single application or a continuous wash orthe spreading of a cream or lotion. Another factor is the proteaseitself since protease activity is substrate-dependent.

5.2 METHODS OF USE OF THE COMPOSITIONS OF THE PRESENT INVENTION

The present invention provides methods for enhancing epidermalexfoliation and/or for enhancing epidermal cell renewal. Such methodscomprise topically administering to an area of a subject's skin aneffective amount of a composition of the present invention, whichcomprises an acid protease enzyme exhibiting proteolytic activity belowabout pH 5.5 and insignificant activity at or above about pH 5.5 and anacidic buffer which lowers the pH of the surface of the skin to belowabout pH 5.5 for a period of time effective to enhance epidermalexfoliation and/or enhance epidermal cell renewal, theacidic bufferbeing subject to neutralization by natural epidermal processes.

The present invention also provides methods for improving the textureand/or appearance of skin. Such methods comprise administering to anarea of a subject's skin an effective amount of a composition of thepresent invention, which comprises an acid protease enzyme exhibitingproteolytic activity below about pH 5.5 and insignificant activity at orabove about pH 5.5 and an acidic buffer which lowers the pH of thesurface of the skin to below about pH 5.5 for a period of time effectiveto improve the texture and/or appearance of skin, the acidic bufferbeing subject to neutralization by natural epidermal processes.

The present invention also provides methods for regulating the effectsof skin atrophy. Such methods comprise administering to an area of asubject's skin an effective amount of a composition of the presentinvention, which comprises an acid protease enzyme exhibitingproteolytic activity below about pH 5.5 and insignificant activity at orabove about pH 5.5 and an acidic buffer which lowers the pH of thesurface of the skin to below pH 5.5 for a period of time effective toregulate the effects of skin atrophy, the acidic buffer being subject toneutralization by natural epidermal processes.

The present invention also provides methods for treating or preventingan abnormal skin condition, disease, or disorder. Such methods compriseadministering to an area of a subject's skin an effective amount of acomposition of the present invention, which comprises an acid proteaseenzyme exhibiting proteolytic activity below about pH 5.5 andinsignificant activity at or above about pH 5.5 and an acidic bufferwhich lowers the pH of the surface of the skin to below pH 5.5 for aperiod of time effective to treat or prevent an abnormal skin condition,disease, or disorder, the acidic buffer being subject to neutralizationby natural epidermal processes. Such condition, disease, or disorderincludes, but is not limited to, dry skin, severe dry skin, dandruff,acne, keratoses, eczema, skin flakiness, pruritus, age spots,lentigines, melasmas, wrinkles (both coarse and fine, caused byintrinsic as well as extrinsic damage), blemished skin, hyperpigmentedskin,hyperkeratotic skin, inflammatory dermatoses, age-relatedskin-changes, skin in need of cleansers, and the effects of skin atrophyand psoriasis.

A preferred method of administering an effective amount of a compositionof the present invention for any of the methods described above on anarea of skin is via topical application. The amount of the acidic bufferand acid protease in the final composition and frequency of topicalapplication to the skin can vary widely, depending upon factors such asthe particular skin disorder, the severity of the skin disorder, thelocation and/or type of the skin involved, the subject's skinsensitivity, and the degree of treatment desired. It is well within thepurview of the skilled artisan to regulate dosages according to thesubject's need. It is suggested as an example that topical applicationrange from about once per week to about 4 or 5 times daily, preferablyfrom about 3 times a week to about 3 times daily, most preferably aboutonce or twice per day. The final composition for topical applicationwill comprise from about 0.001% to about 95%, preferably from about0.01% to about 25%, most preferably from about 0.01% to about 5% of theacid component. The final composition for topical application willcomprise from about 0.001% to about 75%, preferably from about 0.1% toabout 50%, most preferably from about 1.0% to about 5% of the acidprotease component.

Another preferred mode of administering is chronic administration.“Chronic” administration, as used herein, means that the period oftopical application may be over the subject's lifetime, preferably for aperiod of at least about one month, more preferably from about threemonths to about twenty years, more preferably from about six months toabout ten years, more preferably still from about one year to about fiveyears, thereby resulting in the treatment or prevention of the abnormalskin condition, disease, or disorder or in the improvement of thetexture or appearance of the skin, or in the enhancement of epidermalexfoliation and/or epidermal cell renewal or the regulation of theeffects of skin atrophy.

In another embodiment of the invention, the methods described aboveinvolve administering both an effective amount of the compositioncomprising an acidic buffer and an acid protease and an effective amountof one or more of a sunscreening agent, an anti-inflammatory agent, ananti-oxidant/radical scavenging agent, a chelating agent, a retinoid,self-tanning material, and/or a benzofuran derivative to the skinsimultaneously. As used herein, “simultaneous application” or“simultaneously” means administering the agents to the skin at the samesitus on the body at about the same time. Although this can beaccomplished by administering the agents separately to the skin,preferably a composition comprising all the desired agents commingled isadministered to the skin. The amount of sunscreening agent administeredgenerally ranges from about 0.02 mg to about 1.0 mg per cm² skin. Theamount of anti-inflammatory agent administered generally ranges fromabout 0.005 mg to about 0.5 mg, preferably from about 0.01 mg to about0.1 mg per cm² skin. The amount of chelating agent administeredgenerally ranges from about 0.001 mg to about 1.0 mg, preferably fromabout 0.01 mg to about 0.5 mg, more preferably from about 0.05 mg toabout 0.1 mg per cm² skin. The amount of retinoid administered generallyranges from about 0.00001 mg to about 0.02 mg per cm² skin. The amountof benzofuran derivative administered generally ranges from about 0.001mg to about 1.0 mg/cm² skin per application, preferably from about 0.01to about 0.5 mg/cm² skin per application. The amount of the compositioncomprising an acidic buffer and an acid protease administered generallyranges from about 0.001 mg to about 1.0 mg per cm² skin per application,preferably from about 0.01 mg to about 0.5 mg per cm², more preferablyfrom about 0.05 to about 0.25 mg/cm² skin per application, which mayvary upon the severity of the condition to be treated, the sensitivityof the subject's skin, the location of the treatment site on thesubject, and the efficacy of the compounds employed.

6. EXAMPLES

In order to more fully illustrate the nature of the invention and themanner of practicing the same, the following examples are provided,which are not to be construed as limiting the remainder of thedisclosure or the scope of the invention in any way whatsoever.

The following reagents were used in the compositions and methodsdescribed in Section 6.1 through 6.4:

AFP 2000, a fungal-derived acid protease, Solvay Inc., Elkhart, Ind.;algae extract/aloe extract, Active Organics, Inc., Dallas, Tex.;allantoin, Sutton Laboratories, Chatham, N.J.; Arlacel 65, ICISurfactants, Wilmington Del.; aspartic acid, Ajinomoto USA, Inc.,Teaneck, N.J.; BHT, Eastman Kodak, Co., Rochester, N.Y.; butyleneglycol, Hoechst Celanese, Co., Charlotte, N.C.; Carbomer, B.F. Goodrich,Co., Brecksville, Ohio; cetearyl alcohol, Lipo Chemicals, Inc.,Patterson, N.J.; cetyl alcohol, Lipo Chemicals, Inc., Patterson, N.J.;citric acid, Roche Vitamins and Fine Chemicals, Nutley, N.J.; Cosmowax,Croda Chemicals, Ltd., Parsippany, N.J.; cyclomethicone, Dow ChemicalsUSA, Midland, Mich.; dihydroxyacetone, Rona/E. Merck Industries,Hawthorne, N.Y.; dimethicone, Dow Chemicals USA, Midland, Mich.;disodium EDTA, Ciba-Geigy Co., Greensboro, N.C.; DL-Panthenol, RocheVitamins and Fine Chemicals, Nutley, N.J.; ethoxydiglycol, GattefosseS.A., Paris, France; gluconic acid, Sigma Chemicals, St. Louis, Mo.;Glycereth-26, Amerchol Co., Edison, N.J.; glyceral stearate, CrodaChemicals, Ltd., Parsippany, N.J.; lactic acid USP 88%, RitaCorporation, Woodstock, Ill.; lecithin/asiatic acid, Active Organics,Dallas, Tex.; methyl gluceth-20, Amerchol Co., Edison, N.J.;methylparaben, Napp Co., Lodi, N.J.; octyl palmitate, ICI Surfactants,Wilmington, Del.; octyldodecyl neopentanoate, ICI Surfactants,Wilmington, Del.; PEG-75, Lipo Chemicals, Patterson, N.J.;PEG-40stearate, Lipo Chemicals, Inc., Patterson, N.J.; Pepsin 1:15,000NF, American Laboratories, Inc., Omaha, Nebr.; phosphoric acid 85%,Sigma Chemicals, Inc., St. Louis, Mo.; polyacrylamide/C13-14isoparaffin/laureth-7, Seppic, Co., Paris, France; propylene glycol, DowChemicals USA, Midland, Mich.; propylparaben, Napp Co., Lodi, N.J.;retinyl palmitate, Roche Vitamins and Fine Chemicals, Nutley, N.J.;salicylic acid, Kalama Co., Seattle, Wash.; sodiumcarboxyl-methylcellulose 7HXF, Aqualon, Co., Wilmington, Del.; sodiumhyaluronate, Active Organics, Dallas, Tex.; steareth-lo, Lipo Chemicals,Inc., Patterson, N.J.; tocopheryl acetate, Roche Vitamins and FineChemicals, Nutley, N.J.; triethanolamine 99%, BASF Co., Mount Olive,N.J.; and xantham gum, Kelco, San Diego, Calif.

6.1 ENHANCEMENT OF SKIN EXFOLIATION

A composition of the present invention was tested at differingconcentrations of the acid protease pepsin, 1:15,000 NF, AmericanLaboratories, Inc., Omaha, Nebr., and the acidic buffer lactic acid onindividual human volar forearms for the enhancement of skin exfoliationand cell renewal. Twenty subjects between the ages of 30 and 60 yearswere selected and were required to refrain from using any products ontheir volar forearm, except those supplied in conjunction with the testprocedure (which included a synthetic soap) for 5 days before and duringthe test period. None of the subjects: (1) was pregnant or lactating orintended to become pregnant within three months following the test; (2)had one or more known communicable diseases; (3) was involved in anyother clinical study at the time of the test; (4) suffered frompsoriasis, dermatitis, and/or eczema; (5) was taking medicationconsidered likely to interfere with the test results, includingretinoids, corticosteroids, or antibiotics; (6) had taken retinoids,corticosteroids, or antibiotics during the six months prior to the test;and/or (7) had used tobacco products, had sensitive skin, had a skintype deemed unsuitable for the test, or had other characteristicsconsidered likely to make them unsuitable for the test.

Each volar forearm of each subject was patched with four 2×3 cm adhesivebandages, to which had been applied 1-2 gm/cm² of 5% ultra-pure dansylchloride milled into petrolatum. Three of the bandages on each forearmwere used as test sites and the remaining one was used as a control.

The four sites on each forearm of each subject were covered with thedansyl chloride-loaded bandages and were left undisturbed for 24 hours.At the end of the 24 hour period, the bandages were removed, the siteswashed and staining of the sites by dansyl chloride was confirmed byviewing with a long wave UV light source to detect fluorescence by thedansyl chloride. The six non-control dansyl chloride stained test siteson each subject received twice daily topical applications of 1-2 ml/cm²of a randomized selection of test compositions described in Table I.Upon application, the test compositions were rubbed into the skin at thetest sites until the sites were no longer wet. After the dansyl chloridestaining was verified, the test sites and the control sites were leftuncovered and were handled in the same manner except that the test sitesreceived the test applications and the control sites did not receive anytest composition.

TABLE I Test Lactic Acid Acid HUT Sorbic Acid Deionized Composi- (100%)Protease^(a) units/g* preservative Water tion No. % by wt. % by wt. 10⁶% by wt. % by wt. 1 0.00 0.00 0.0 0.05 99.95 2 1.50 0.00 0.0 0.05 98.453 1.50 0.02  0.02 0.05 98.43 4 1.50 0.20 0.2 0.05 98.25 5 1.50 1.00 1.00.05 97.45 6 1.50 2.00 2.0 0.05 96.45 7 1.50 4.00 4.0 0.05 94.45 8 3.000.00 0.0 0.05 96.95 9 3.00 0.02  0.02 0.05 96.93 10  3.00 0.20 0.2 0.0596.75 11  3.00 1.00 1.0 0.05 95.95 12  3.00 2.00 2.0 0.05 94.95 13  3.004.00 4.0 0.05 92.95 ^(a)The acid protease employed was Pepsin, 1:15,000,NF, American Laboratories, Inc., Omaha NE.

Exfoliation/keratolysis of the stratum corneum was determined byvisualizing the dansyl chloride stains daily under a long wave UV lightsource to measure stain removal. The percent increase inexfoliation/keratolysis and accompanying cell renewal of the stratumcorneum was calculated by the following formula:${\% \quad {Increase}} = {\frac{\begin{matrix}\left( {{{days}\quad {for}\quad {removal}\quad {of}\quad {untreated}\quad {area}} -} \right. \\\left. {{days}\quad {for}\quad {removal}\quad {of}\quad {treated}\quad {area}} \right)\end{matrix}}{{days}\quad {for}\quad {removal}\quad {of}\quad {untreated}\quad {area}} \times 100}$

TABLE II Pepsin Effect (% Effect of Increase Test Composition overLactic Composi- Lactic Acid HUT (% Increase Acid tion Acid Protease^(a)units/ over Control Control No. Conc. (%) Conc. (%) g* 10⁶ Value)Values) 1 0.00 0.00 0.0 0 (Control)  0 2 1.50 0.00 0.0 14 0 (Control) 31.50 0.02  0.02 14  0 4 1.50 0.20 0.2 17 21 5 1.50 1.00 1.0 25 79 6 1.502.00 2.0 28 100  7 1.50 4.00 4.0 33 136  8 3.00 0.00 0.0 18 0 (Control)9 3.00 0.02  0.02 18  0 10  3.00 0.20 0.2 23 28 11  3.00 1.00 1.0 31 7212  3.00 2.00 2.0 33 83 13  3.00 4.00 4.0 36 100  ^(a)The acid proteaseemployed was Pepsin, 1:15,000, NF, American Laboratories, Inc., OmahaNE.

The data collected from the test are summarized in Table II and in FIGS.1 and 2. As seen in Table II, column 5, acidic buffer lactic acid hadsome positive keratolytic/cell renewal effects alone [compare testcomposition 2 and 8 to the control (test composition 1)] atconcentrations of 1.5% and 3.0% by weight. These positivekeratolytic/cell renewal effects are significantly enhanced in thepresence of the acid protease pepsin, (see test compositions 3-7 and9-13). It is also apparent that the enhancements are dose dependent,i.e., stratum corneum exfoliation/cell renewal rates increase as theconcentration of the acid protease increases, see column of Table II.FIG. 1 presents the data in column 5 graphically.

Column 6 of Table II reflects the percent increase in exfoliation/cellrenewal rates given by various concentrations of the acid proteasepepsin over that given by lactic acid alone at 1.5% and 3.0% by weightconcentration. The data in column 6 is presented graphically in FIG. 2.It is apparent that, although the acid protease effect is significant atboth lactic acid concentrations employed, the greater percentenhancements are given by the acid protease pepsin at a lactic acidconcentration of 1.5% by weight.

A low level of redness and swelling was observed on the skin of some ofthe test subjects, approximately 75% of the skin test sites at a 3% byweight lactic acid concentration, but very little was observed at a 1.5%by weight lactic acid concentration. Furthermore, this finding waslargely independent of the presence of pepsin. Thus, significantexfoliation/cell renewal is accomplished by employing an acidic bufferat a low, non-irritating level in the presence of an appropriateconcentration of an acid protease. In contrast to proteases which areactive in the physiological pH range, acid proteases, as defined herein,applied to the skin maintain their proteolytic activity only until theskin's own natural processes raise the pH of the skin's surface to itsnormal value of approximately 5.5.

6.2 COMPOSITIONS FOR USE IN THE METHODS OF THE PRESENT INVENTION

The following -Tables recite formulations of different examples ofcompositions of the present invention. All of the following compositionswere adjusted to a pH of about 3.0 to 3.2 with, for example, dilutephosphoric acid or sodium hydroxide, NaOH.

TABLE III WASH AP-01 PERCENT BY WEIGHT ASPARTIC ACID 1.50 DEIONIZEDWATER 82.50 METHYLPARABEN 0.20 PEG 75 1.50 DISODIUM EDTA 0.05 ALLANTOIN0.25 GLYCERETH 26 1.00 ETHOXYDIGLYCOL 4.00 PROPYLENE GLYCOL 4.00 AFP2000 5.00 TOTALS 100.00

TABLE IV WASH AP-02 PERCENT BY WEIGHT PHOSPHORIC ACID 85% 1.20 DEIONIZEDWATER 86.80 METHYLPARABEN 0.20 PEG 75 1.50 DISODIUM EDTA 0.05 ALLANTOIN0.25 GLYCERETH 26 1.00 ETHOXYDIGLYCOL 4.00 PROPYLENE GLYCOL 4.00 PEPSIN1,000 HUT UNITS/MG 1.00 TOTALS 100.00

TABLE V Serum AP-03 PERCENT BY WEIGHT SALICYLIC ACID 0.50TRIETHANOLAMINE 99% 1.00 LACTIC ACID USP 88% 1.20 DEIONIZED WATER 82.70SODIUM CARBOXY-METHYCELLULOSE 0.60 7HXF METHYLPARABEN 0.20 PEG 75 1.50DISODIUM EDTA 0.05 ALLANTOIN 0.25 METHYL GLUCETH-20 1.00 GLYCERETH 261.00 ETHOXYDIGLYCOL 4.00 PROPYLENE GLYCOL 4.00 LECITHIN/ ASIATIC ACID1.00 PEPSIN 1,000 HUT UNITS/MG 1.00 TOTALS 100.00

TABLE VI Serum AP-04 PERCENT BY WEIGHT GLUCONIC ACID 1.50 DEIONIZEDWATER 84.08 METHYLPARABEN 0.02 SODIUM CARBOXY-METHYCELLULOSE 0.60 7HXFPEG 75 1.50 DISODIUM EDTA 0.05 ALLANTOIN 0.25 METHYL GLUCETH-20 1.00GLYCERETH 26 1.00 ETHOXYDIGLYCOL 4.00 PROPYLENE GLYCOL 4.00 LECITHIN/ASIATIC ACID 1.00 PEPSIN 1,000 HUT UNITS/MG 1.00 TOTALS 100.00

TABLE VII Pepsin Lotion AP-05 PERCENT BY WEIGHT DEIONIZED WATER 64.75XANTHAN GUM 0.10 DISODIUM EDTA 0.10 ALLANTOIN 0.50 ALGAE EXTRACT (and)ALOE EXTRACT 6.00 METHYLPARABEN 0.20 DL-PANTHENOL 0.50 ETHOXYDIGYCOL4.00 BUTYLENE GYCOL 5.00 CETYL ALCOHOL 2.00 GLYCERYL STEARATE 2.00 OCTYLPALMITATE 4.00 CYCLOMETHICONE 2.00 PROPYLPARABEN 0.10 BHT 0.05POLYACRYLAMIDE (and) C 13-14 1.50 ISOPARAFFIN (and) LAURETH-7 SODIUMHYALURONATE 3.00 RETINYL PALMITATE 0.10 TOCOPHERYL ACETATE 0.10 PEPSIN1,000 HUT UNITS/MG 1.00 CITRIC ACID 3.00 TOTALS 100.00

TABLE VIII Renewal Gel AP-06 PERCENT BY WEIGHT DEIONIZED WATER 89.60CARBOMER 1.50 SALICYLIC ACID 0.50 LACTIC ACID USP 88% 1.20 PROPYLENEGLYCOL 6.00 PEPSIN 1,000 HUT UNITS/MG 1.00 METHYLPARABEN 0.15PROPYLPARABEN 0.05 TOTALS 100.00

TABLE IX Renewal Gel AP-07 PERCENT BY WEIGHT DEIONIZED WATER 85.10POLYACRYLAMIDE (and) C13-14 4.00 ISOPARAFFIN (and) LAURETH-7 SALICYLICACID 0.50 LACTIC ACID USP 88% 1.20 BUTYLENE GLYCOL 8.00 PEPSIN 1,000 HUTUNITS/MG 1.00 METHYLPARABEN 0.15 PROPYLPARABEN 0.05 TOTALS 100.00

TABLE X Self-Tanning Lotion AP-08 PERCENT BY WEIGHT DEIONIZED WATER48.15 XANTHAN GUM 0.75 PROPYLENE GLYCOL 5.00 METHYLPARABEN 0.20PROPYLPARABEN 0.10 PEPSIN 1,000 HUT UNITS/MG 2.00 STEARETH-10 0.70ARLACEL 1651 1.20 PEG-40 STEARATE 0.40 COSMOWAX J2 1.00 CETEARYL ALCOHOL1.50 CYCLOMETHICONE 5.00 DIMETHICONE 0.50 OCTYLDODECYL NEOPENTANOATE28.50 DIHYDROXYACETONE 5.00 TOTALS 100.00

6.3 STABILIZATION COMPOSITION

The following composition at pH 3.0 retains at least 90% of its acidprotease activity over a period of at least-three months, during whichthe composition was stored at 37° C.

% by weight Cyclomethicone 5.0 Cetyl Alcohol 1.5 Arlacel 165 1.5Dimethicone 1.0 Sepigel 305 3.0 Phenoxyethanol 1.0 EDTA, disodium 0.1Butylene Glycol 10.0  Citric acid/Sodium Citrate 3.0 Lactic acid (100%)1.5 Sorbic acid  0.05 Lactose 1.0 Pepsin; 1.0 1000 HUT Units/mg H₂O70.35

6.4 PROTEOLYTIC ACTIVITY AT VARYING pH LEVELS

Two acid protease enzymes were tested over a range of pH values todetermine if they have proteolytic activity at a pH less than about pH5.5 and whether the activity decreases to less than about 10% comparedto its activity at the pH optimum. Proteolytic activity was measured bydetermining the proteolytic effect on hemoglobin using the HUT assay asdescribed below. The two acid proteases were pepsin 1:15,000 NF, atabout 1000 HUT units/mg, supplied by American Laboratories, Inc., OmahaNebr., and AFP 2000, a fungally-derived acid protease supplied bySolvay, Inc., Elkhart, Ind., at about 100 HUT units/mg. The assay wasperformed first by preparing stock solutions HEMOGLOBIN SUBSTRATE andTCA STOCK. The hemoglobin substrate was made by dissolving 2 g of bovinehemoglobin, Sigma Chemicals, Inc., St. Louis, Mo., into 80 ml distilledwater. This solution was then titrated to the correct pH, either, pH 2,3.5, 5.5 or 6.0. For pH values between 1 and 3, 1.8 g of phosphoric acidwas dissolved for a final concentration of 100 mM and the pH wasadjusted with 50% sodium hydroxide or 50% hydrochloric acid. For pHvalues between 3 and 6, 2 g of citric acid was dissolved for a finalconcentration of 100 mM and the pH was adjusted with 50% sodiumhydroxide or hydrochloric acid. The solutions were brought to a finalvolume of 100 ml by adding distilled water. The substrates withdiffering pH values were then incubated at 30° C. for 20 minutes and'subsequently filtered through glass wool. The TCA stock solution wasprepared by dissolving 5 g of trichloroacetic acid into 100 ml distilledwater.

The enzyme standards were then prepared by dissolving mg of pepsin in mldistilled water and by dissolving 100 mg of AFP 2000 and 300 mg citricacid in 8 ml distilled water. This solution was then adjusted to a pH ofabout 3.5 with 6M HCl of 50% sodium hydroxide and the final volume wasbrought to ml by the addition of distilled water.

For each of the samples, four tubes were marked, “B”, “T1”, “T2” and“T3”, and 4 ml of the hemoglobin substrate solution was added to eachtube, which were then placed at 37° C. 100 μl of the enzyme standard wasadded to each “T” labeled tube, i.e., T1, T2 and T3. The tubes were thenincubated for minutes at 37° C. for pepsin and minutes for AFP 2000. 10ml of the TCA stock solution was then added to all the tubes, and 100 μlof enzyme was added to tube “B”. The tubes were then centrifuged for oneminute at 1000 rpm. The sample is each tube was filtered through asyringe filter into a quartz cuvette and the absorbance at 280 nm wastaken. The background absorbance was deducted from each of the “T”'sabsorbance and the individual “T1”, “T2” and “T3” absorbencies wereaveraged for each enzyme at each pH tested.

FIG. 3 demonstrates that both of the enzymes were optimally active at pH2.0. Further, both enzymes had less than 10% activity at pH 5.5 andgreater as compared to the optimal level at pH 2.0. This assay clearlydemonstrates that these enzymes-are enzymes which can be used in thepresent invention. Further, by using such an assay, it may be determinedwhich additional enzymes are suitable for use in the present invention.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed since these embodiments areintended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

A number of references are cited herein, the entire disclosures of whichare incorporated herein, in their entirety, by reference.

What is claimed is:
 1. A method for treating at least one of an abnormalskin condition, disease, disorder and for regulating the effects of skinatrophy and for improving the texture or appearance of the skincomprising the step of: topically administering to a subject on an areaof skin an effective amount of a composition comprising: (1) an acidprotease which is enzymatically active below about pH 5.5; and (2) anacidic buffer which lowers the surface pH of the skin to below about pH5.5 for a period of time effective for the treatment of at least onecondition, disease, and disorder, and for regulating at least one of theeffects of skin atrophy and for improving the texture and appearance ofthe skin, the acidic buffer being subject to neutralization by naturalepidermal processes, such that the surface pH of the skin returns toabout pH 5.5.
 2. The method according to claim 1 wherein the acidicbuffer further comprises at least one of a pharmaceutically andcosmetically acceptable carrier, vehicle excipient.
 3. The methodaccording to claim 1 wherein the acid protease is selected from thegroup consisting of fungal proteases, bacterial proteases, mammalianproteases and mixtures thereof.
 4. The method according to claim 1wherein the acid protease is selected from the group consisting ofpepsin, cathepsin, human urinary acid protease, rhizopuspepsin,penicillopepsin, endothiapepsin or mixtures thereof.
 5. The methodaccording to claim 1 wherein the acid protease is pepsin.
 6. The methodaccording to claim 1 wherein the acid component of the acidic buffer isselected from the group consisting of inorganic acids, organic acids ormixtures thereof.
 7. The method according to claim 6 wherein theinorganic acid is selected from the group consisting of phosphoric acid,pyrophosphoric acid, triphosphoric acid, polyphosphoric acid, sodiumbisulfate, potassium bisulfate or mixtures thereof.
 8. The methodaccording to claim 6 wherein the organic acid is selected from the groupconsisting of lactic acid, citric acid, sorbic acid, glycolic acid,malic acid, gluconic acid or mixtures thereof.
 9. The method accordingto claim 2 wherein the at least one pharmaceutically, cosmeticallyacceptable carrier, vehicle and excipient component of the acidic bufferis selected from the group consisting of lotions, tinctures, creams,emulsions, gels, ointments, water, water-workable cream, polyvinylalcohol, hydroxyethyl cellulose, cellulose, hydrophilic acrylic polymer,emollients, skin moisturizing components, enzyme stabilizers, glycerol,surfactants, preservatives, hydrophilic thickening agents used inpharmaceutical formulations and mixtures thereof.
 10. The methodaccording to claim 1 wherein the acid protease is present in an amountof about 0.001% to about 75.0% by weight of the final composition. 11.The method according to claim 1 wherein the acid protease is present inan amount of about 0.1% to about 50.0% by weight of the finalcomposition.
 12. The method according to claim 1 wherein the acidprotease is present in an amount of about 1.0% to about 5.0% by weightof the final composition.
 13. The method according to claim 1 whereinthe acid protease has a total specific activity of about 1.0 to about5000 HUT units/mg.
 14. The method according to claim 1 wherein the acidprotease has a total specific activity of about 500 to about 3000 HUTunits/mg.
 15. The method according to claim 1 wherein the acid proteasehas a total specific activity of about 500 to about 1500 HUT units/mg.16. The method according to claim 1 wherein the acid component of theacidic buffer is present in an amount of about 0.001% to about 95.0% byweight of the final composition.
 17. The method according to claim 1wherein the acid component of the acidic buffer is present in an amountof about 0.01% to about 25.0% by weight of the final composition. 18.The method according to claim 1 wherein the acid component of the acidicbuffer is present in an of about 1.0% to about 5.0% by weight of thefinal composition.
 19. The method according to claim 1 wherein thesurface pH of the skin is below about pH 5.5 for about 5 minutes toabout 4 hours.
 20. The method according to claim 1, wherein the surfacepH of the skin is below about pH 5.5 for about 30 minutes to about 2hours.
 21. The method according to claim 1 wherein the pH of the skin isbelow about pH 5.5 to about 1 hour.
 22. The method-according to claim 1wherein abnormal skin condition, disease or disorder is selected fromthe group consisting of dry skin, severe dry skin, dandruff, acne,keratoses, psoriasis, eczema, skin flakiness, pruritus, age spots,lentigines, melasmas, wrinkles, warts, blemished skin, hyperpigmentedskin, hyperkeratotic skin, inflammatory dermatoses, age-related skinchanges, and psoriasis.